Shengmai Injection Alleviates H2O2‐induced Oxidative Stress through Activation of AKT and Inhibition of ERK Pathways in Neonatal Rat Cardiomyocytes
Shengmai Injection Alleviates H2O2‐induced Oxidative Stress through Activation of AKT and Inhibition of ERK Pathways in Neonatal Rat Cardiomyocytes
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生脉注射液通过激活 AKT 和抑制 ERK 通路减轻 H2O2 诱导的新生大鼠心肌细胞氧化应激
DOI：
10.1016/j.jep.2019.01.001
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发表时间：
--
影响因子：
5.4
通讯作者：
Shengyu Hua
中科院分区：
文献类型：
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作者：
Jinqiang Zhu;Qiaofeng Ye;Shixin Xu;Yan-xu Chang;Xuan Liu;Yan Ma;Yan Zhu;Shengyu Hua
生脉注射液是《中华人民共和国中国药典》(2015版)正式收录的一种经典中药，中国长期以来一直用于治疗心力衰竭。本研究旨在评价参麦注射液减轻过氧化氢氧化应激的作用及其机制。材料与方法采用原代培养的新生大鼠心肌细胞，建立过氧化氢诱导的心肌细胞氧化应激模型。采用CCK-8细胞活力检测试剂盒和乳酸脱氢酶细胞毒性检测试剂盒检测CCK8细胞存活率和乳酸脱氢酶细胞毒作用，以确定该模型的安全剂量和最低有效剂量。用CM-H_2DCFDA荧光探针检测H_2O_2诱导氧化应激心肌细胞的ROS水平。观察氧化应激过程中心肌细胞内NAD(P)H水平的变化。用Fura-2/AM和Rhod-2-AM荧光探针检测氧化应激过程中心肌细胞内钙离子和钙离子的含量。用Annexin V-FITC/PI双染法检测氧化应激心肌细胞的凋亡情况。应用Annexin V-FITC/PI双染凋亡检测试剂盒检测氧化应激后心肌细胞的凋亡情况。采用定量聚合酶链式反应(RT-PCR)检测抗氧化酶过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽还原酶(GSR)的表达。用Western印迹法检测AKT和ERK1/2的磷酸化情况。结果参麦注射液能显著减轻氧化应激诱导的新生大鼠心肌细胞增殖停滞和细胞凋亡。此外，参麦还能减少细胞内活性氧(ROS)、烟酰胺腺嘌呤二核苷酸(NADH)和丙二醛(MDA)的产生，减轻H_2O_2引起的细胞质和线粒体Ca~(2+)超载。参麦还能恢复H_2O_2抑制的SOD、GSR和CAT的mRNA表达和活性。机制上，参麦可上调过氧化氢处理的心肌细胞内AKT的磷酸化，下调ERK1/2的磷酸化。AKT磷酸化抑制剂LY294002可抑制SMI对心肌细胞的保护作用，而ERK1/2磷酸化抑制剂PD98059可增强SMI的保护作用。结论SMI可能通过AKT和ERK1/2途径减轻心肌细胞氧化应激损伤，有望成为治疗氧化应激损伤的可注射性中药。
Ethnopharmacological relevanceShengmai injection (SMI) is a classical traditional Chinese medicine (TCM) officially recorded in Pharmacopoeia of the People's Republic of China (version 2015) and has long been used to treat heart failure in China. However scientific evidence for the anti-oxidative stress potential of SMI used in traditional medicine is lacking.Aim of studyThe present study aimed to evaluate the efficacy of SMI in alleviating H2O2‑induced Oxidative Stress the underlying mechanismsMaterials and methodsH2O2-induced oxidative stress model of cardiomyocytes was established with primary cultured neonatal rat cardiomyocytes. CCK8 cell viability assay and lacatate dehydrogenase cytotoxicity assay were performed to ensure the safety dose and lowest effective dose for the mode employing CCK-8 cell viability assay kit and lactate dehydrogenase cytotoxicity assay kit. ROS levels were determined using CM-H2DCFDA fluorescent probe in cardiomyocytes with H2O2-induced oxidative stress. The change of NAD(P)H level in cardiomyocytes was evaluated during the process of oxidative stress. The content of myocardial cytosolic Ca2+and Ca2+was determined using Fura-2/AM and Rhod 2-AM fluorescent probe in mitochondrial in the process of oxidative stress. Annexin V-FITC/PI double staining was applied to examine the apoptotic cells in cardiomyocytes with oxidative stress. To identify the apoptosis after oxidative stress myocardial cells with the application of Annexin V-FITC/PI double staining apoptosis detection kit. Quantitative polymerase chain reaction (RT-PCR) was applied to measure the expression of antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSR). Western blot was performed to observe the phosphorylation of AKT and ERK1/2.ResultsSMI was shown to significantly attenuate oxidative stress-induced cell proliferation arrest and apoptosis in neonatal rat cardiomyocytes. In addition, SMI treatment could decrease the production of reactive oxygen species (ROS), nicotinamide adenine dinucleotide (NADH) and malondialdehyde (MDA), and reduce the overloads of cytoplasmic Ca2+and mitochondrial Ca2+induced by H2O2. SMI could also restore the mRNA expression and activities of SOD, GSR, and CAT suppressed by H2O2. Mechanistically, SMI upregulated intracellular AKT phosphorylation and downregulate ERK1/2 phosphorylation in H2O2-treated cardiomyocytes. Pretreatment with LY294002, an AKT phosphorylation inhibitor, suppressed the protective role of SMI in cardiomyocytes, while pretreatment with PD98059, an ERK1/2 phosphorylation inhibitor, enhanced the effect of SMI.ConclusionsIn conclusion, SMI may attenuate oxidative stress-induced damage in cardiomyocytes potentially through the AKT and ERK1/2 pathway and can function as a promising injectable traditional Chinese medicine to treat oxidative stress-induced injury.